Synthesis gas (syngas) is a mixture of carbon monoxide (CO) gas, carbon dioxide (CO2) gas, and hydrogen (H2) gas, and other volatile gases such as CH4, N2, NH3, H2S and other trace gases. Syngas is produced by gasification of various organic materials including biomass, organic waste, coal, petroleum, plastics, or other carbon containing materials, or reformed natural gas.
Acetogenic Clostridial microorganisms grown in an atmosphere containing syngas are capable of absorbing the syngas components CO, CO2, and H2 and producing aliphatic C2-C6 alcohols and aliphatic C2-C6 organic acids. These syngas components activate Wood-Ljungdahl metabolic pathway 100, as shown in FIG. 1, which leads to the formation of acetyl coenzyme A 102, a key intermediate in the pathway. Under autotrophic fermentation conditions, the enzymes activating Wood-Ljundahl pathway 200, as shown in FIG. 2, are carbon monoxide dehydrogenase (CODH) 104 and hydrogenase (H2ase) 106. These enzymes capture the electrons from the CO and H2 in the syngas and transfer them to ferredoxin 108, an iron-sulfur (FeS) electron carrier protein. Ferredoxin 108 is the main electron carrier in metabolic pathway 100 in acetogenic Clostridia, primarily because the redox potential during syngas fermentation is very low (usually between −400 and −500 mV). Upon electron transfer, ferredoxin 108 changes its electronic state from Fe3+ to Fe2+. Ferredoxin-bound electrons are then transferred to cofactors NAD+ 110 and NADP+ 112 through the activity of ferredoxin oxidoreductases 114 (FORs). The reduced nucleotide cofactors (NAD+ and NADP+) are used for the generation of intermediate compounds in Wood-Ljungdahl pathway 100 leading to acetyl-CoA 102 formation.
The FOR-mediated ferredoxin reduction reaction additionally feeds an Rnf complex that maintains a proton-motive force (PMF) to generate ATP through interconnectivity of the electron-motive force (EMF) with the PMF since under autotrophic growth conditions homoacetogenic cells are generating net ATP through a PMF via an F1F0 type ATP synthase consisting of seven genes (see Table 1, Gene ID Nos. 50-57). The net ATP generated through operation of the Rnf complex is then consumed for cell growth or maintenance.
Acetyl-CoA 102 formation through Wood-Ljungdahl pathway 100 is shown in greater detail in FIG. 2. Either CO2 202 or CO 208 provide substrates for the pathway. The carbon from CO2 202 is reduced to a methyl group through successive reductions first to formate, by formate dehydrogenase (FDH) enzyme 204, and then is further reduced to methyl tetrahydrofolate intermediate 206. The carbon from CO2 208 is reduced to carbonyl group 210 by carbon monoxide dehydrogenase (CODH) 104 through a second branch of the pathway. The two carbon moieties are then condensed to acetyl-CoA 102 through the action of acetyl-CoA synthase (ACS) 212, which is part of a carbon monoxide dehydrogenase (CODH/ACS) complex. Acetyl-CoA 102 is the central metabolite in the production of C2-C6 alcohols and acids in acetogenic Clostridia. 
Ethanol production from Acetyl CoA 102 is achieved via one of two possible paths. Aldehyde dehydrogenase facilitates the production of acetaldehyde, which is then reduced to ethanol by the action of primary alcohol dehydrogenases. In the alternative, in some homoacetogenic microorganisms, a bifunctional NADPH-dependent ADH/acetyl CoA reductase (“AR”) thioesterase facilitates the production of ethanol directly from acetyl CoA.
Wood-Ljungdahl pathway 100 is neutral with respect to ATP production when acetate 214 is produced (FIG. 2). When ethanol 216 is produced, one ATP is consumed in a step involving the reduction of methylene tetrahydrafolate to methyl tetrahydrofolate 206 by a reductase, and the process is therefore net negative by one ATP. The pathway is balanced when acetyl-PO4 218 is converted to acetate 214.
Acetogenic Clostridia organisms generate cellular energy by ion gradient-driven phosphorylation. When grown in a CO atmosphere, a transmembrane electrical and chemical potential is generated and used to synthesize ATP from ADP. Enzymes mediating the process include hydrogenase, NADH dehydrogenases, carbon monoxide dehydrogenase, and methylene tetrahydrofolate reductase. Membrane carriers that have been shown to be likely involved in the ATP generation steps include quinone, menaquinone, and cytochromes.
The acetogenic Clostridia produce a mixture of C2-C6 alcohols and acids, such as ethanol, n-butanol, hexanol, acetic acid, and butyric acid, that are of commercial interest through Wood-Ljungdahl pathway 100. For example, acetate and ethanol are produced by C. ragsdalei in variable proportions depending in part on fermentation conditions. However, the cost of producing the desired product, an alcohol such as ethanol, for example, can be lowered significantly if the production is maximized by reducing or eliminating production of the corresponding acid, in this example acetate. It is therefore desirable to metabolically engineer acetogenic Clostridia for improved production of selected C2-C6 alcohols or acids through Wood-Ljungdahl pathway 100 by modulating enzymatic activities of key enzymes in the pathway.
Acetogenesis as described above is a general metabolic trait that is not phylogenetically conserved. Therefore, production of liquid fuels via biocatalyst is the result of a unique collection of genes and functional protein activities that are expressed when grown in the presence of syngas under desirable growth conditions. Not all organisms that have the Wood-Ljungdahl pathway make ethanol, since some lack alcohol dehydrogenases or other genes to convert acetate to ethanol. Thus, the ability to convert syngas components to high ethanol titers is embodied in the unique collection of conserved genes described below.